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目的克隆、表达细粒棘球绦虫基底膜特异性硫酸乙酰肝素聚糖核心蛋白(H3),并评价其检测囊型包虫病的效果。方法将从细粒棘球绦虫原头节cDNA文库中免疫筛选的H3基因,克隆入pGEX-4T表达载体,将重组质粒转化大肠杆菌BL21细胞,用异丙基硫代半乳糖苷(IPTG)进行诱导表达,用亲和层析法纯化重组蛋白,用ELISA方法检测囊型包虫病患者血清、其他寄生虫病患者血清及健康人血清,评价其检测效能,并与本课题组制备的细粒棘球蚴包囊液粗抗原(Hydatid cyst fluid,HCF)和重组Ag B8/2进行比较。结果成功构建细粒棘球蚴pGEX-4T-H3重组质粒,并在原核细胞中成功表达。重组H3抗原、纯化HCF和rAgB8/2检测囊型包虫病患者血清的敏感度分别为84.0%(68/81)、90.1%(73/81)、77.8%(63/81),3者差异无统计学意义(χ~2=4.58,P>0.05)。重组H3抗原与泡型包虫病患者、囊虫病患者及血吸虫病患者血清分别存在63.3%(19/30)、16.7%(5/30)和5.0%(1/20)的交叉反应,与50份健康者血清无交叉反应;H3检测总特异度为80.8%(105/130),H3、纯化HCF[71.5%(93/130)]和rAgB8/2[82.3%(107/130)]特异度间差异无统计学意义(χ~2=5.71,P>0.05)。结论重组H3抗原在囊型包虫病诊断上具有潜在的应用价值。
Objective To clone and express the basal membrane-specific heparan sulfate proteoglycan (H3) of Echinococcus granulosus and evaluate its efficacy in detection of cystic echinococcosis. Methods The H3 gene was screened from the original cDNA library of Echinococcus granulosus and cloned into pGEX-4T expression vector. The recombinant plasmid was transformed into E. coli BL21 cells and treated with isopropylthiogalactoside (IPTG) The recombinant protein was purified by affinity chromatography. Serum of patients with cystic echinococcosis, serum of other parasitic diseases and healthy human serum were detected by ELISA, and the detection efficiency was evaluated. Hydatid cyst fluid (HCF) and recombinant Ag B8 / 2 were compared. Results The recombinant plasmid pGEX-4T-H3 of Echinococcus granulosus was successfully constructed and successfully expressed in prokaryotic cells. The sensitivities of recombinant H3 antigen, purified HCF and rAgB8 / 2 serum in patients with cystic echinococcosis were 84.0% (68/81), 90.1% (73/81) and 77.8% (63/81), respectively No statistical significance (χ ~ 2 = 4.58, P> 0.05). There were 63.3% (19/30), 16.7% (5/30) and 5.0% (1/20) cross-reactivities in serums of patients with cytogenetics and patients with cysticercosis and schistosomiasis respectively. There was no cross-reaction in 50 healthy volunteers; the specificity of H3 was 80.8% (105/130), H3, HCF [71.5% (93/130)], and rAgB8 / 2 There was no significant difference between degrees (χ ~ 2 = 5.71, P> 0.05). Conclusion The recombinant H3 antigen has potential application in the diagnosis of cystic echinococcosis.